Presently, there is a general burden need for fast, sensitive and quantitative diagnostic technologies. Such ones are suitable for wide market areas including public health, research, farming, environmental care, veterinary medicine, and certain industrial production areas. Improved sensitivity, speed, robustness, stability, and decreased cost per analysis are factors, which after being accomplished in diagnostic technologies, can find applications in quite new areas.
Very high sensitivity can be obtained with certain diagnostics instruments, but they are too expensive. On the other hand, certain methods can be inexpensive enough, exemplified by immunochromatography, but they are not applicable to certain needs of the market. Any technology, wherein a set of such demands are met, will have an important place in the future diagnostics and a huge market potential.
There is a number of different analytical principles in practical use in diagnostics: for example, assays based on radioactivity, enzyme-linked immunosorbent assay (ELISA), colorimetric assays, and assays based on fluorescence, and chemiluminescence including electroluminescence (EL). Here, EL is considered to be equivalent to any luminescence that is electrically induced. Thus, electroluminescence is considered to include both anodic as well as hot electron-induced (cathodic) electrochemiluminescence (ECL). The hot electron-induced ECL (HECL) is described in detail in U.S. Pat. No. 6,251,690, Kulmala S., et al. Each of these techniques has their role as regards to the integral of sensitivity, robustness, stability, speed, and price. The differences between the techniques reflect the function of physical limitations or advantages of the methods. For example, a drawback of the application based on radioactive compound is the decay of the label within a period of time and the extra cost of radioactive waste from both the safety and environmental viewpoint. The application of the most sensitive assays on diagnostics is limited by the complicated nature of the tests and instruments, and only experts can perform the assays. The complexity of the assay is generally directly proportional to the price of the instrument and/or the test. In the context of complex instruments, it could be mentioned the anodic electrochemiluminescence techniques are now becoming more and more popular: the instrument is a complicated laboratory robot, the handling of which needs expertise and where the measuring process involves repeated washes and preparative steps. They are factors that increase the cost of the analyses as well as increase the amount of waste and therefore will make this method impossible for the needs of small laboratories, doctors offices etc. (bedside or point of care analytics).
Commercially beneficial methods are based on the principle that the substances to be analyzed are identified and measured in mixtures by so-called label substances. In the measurements based on unique properties of biological molecules, as in immunochemical assays, the analyte to be measured (X) can be selectively sorbed from a mixture of molecules to solid-phase bound antibodies and then the bound molecules are measured with another labeled antibody selectively binding to (X). The label substances can be radioactive isotopes, enzymes, light absorbing, fluorescent or phosphorescent molecules, certain metal chelates etc., which are linked covalently to the antibody. Alternatively, the purified (X) can be marked and the amount of unknown unlabeled sample (X) can be measured by a competition reaction. The assays for DNA and RNA can be also based on the selective binding (bioaffinity). Also many other chemical and biochemical analyses can be carried out by the same principles. In order to decrease the cost and/or increase the measuring accuracy, there is presently a tendency to measure several different parameters at the same time in the sample. One possibility is to use labels showing fluorescence or phosphorescence at different wavelength or possessing different luminescence lifetimes. Different measuring principles and strategies, which can be used in immunodiagnostics, have been described in the book The Immunoassay Handbook, Edited by David Wild, Stockton Press Ltd., New York, 1994, on pages 1-618.
It is known in the prior art that organic substances and metal chelates are beneficial as label substances and that they can be excited by light or in some cases electrochemically to produce luminescence specific to the label. These methods are particularly sensitive and are well suitable for many type of bioaffinity assays. However, because the measured concentrations are extremely low, there are also case-dependent difficulties; the use of fluorescence can be disturbed, among other things, by Tyndall, Rayleigh and Raman scattering. When measuring biological substances, there is, almost without exception, after the excitation pulse, a fast-discharging high background fluorescence. Phosphorescence in the solution phase can be utilized mostly only with chelates between lanthanide ions and specially synthesized organic molecules. The drawback of the excitation techniques with the photoluminescent labels is the complexity of the instruments and the high price of the sensitive optical components.
In general, the advantage of ECL is the low price of the electrical excitation components and simpler optics. Compared to photoluminescence, several drawbacks can be avoided. Traditional anodic electrochemiluminescence with inert metal electrodes can be carried out with organic luminophores by a relative simple instrument in non-aqueous solvents. However, in bioaffinity assays, where the biggest commercial expectations are concentrated to, water solutions are applied. Biological samples are taken nearly always in non-organic solutions and therefore the measuring system should work in aqueous or at least in micellar water solutions. Only a very limited number of transition metal chelates work as ECL-labels in anodic ECL in water or micellar solutions.
Thus far, the commercially most important analytical chemical application of the anodic ECL is the method using derivatives of Ru(bpy)32+-chelate, where the detection of the label occurs in micellar phase. As known from textbooks, micellar mixtures are always prone to different disturbing effects due to the uncontrolled complexity of the micellar equilibria. Similar systems can be used also in very small detection cells in capillary electrophoresis systems (A. Aurora et al., Anal. Comm. 34 (1997) 303-395.). HECL which does not depend on micelles has many crucial advantages over the anodic ECL. It can be applied both to immuno- and DNA hybridization methods (see, Blackburn, G., et al., 1991, Clin. Chem. 37: 1534-1539; Kenten, J., et al. 1992, Clin. Chem. 33: 873-879). The immunoassays and DNA or RNA probe applications by Roche Diagnostics Ltd. exploit magnetic particles by which the label substance is brought onto golden working electrode (Massey; Richard J., et al. U.S. Pat. No. 5,746,974; Leland; Jonathan K., et al. U.S. Pat. No. 5,705,402). The reproducible handling of magnetic latex particles is however in many ways difficult, therefore this method is useful only in expensive laboratory robots (e.g. Elecsys 1010 and 2010) having a complicated and precise liquid o handling system. In addition, the permanent massive golden work electrode needs long cleaning and pretreatment between each analysis (Elecsys Service Manual, p. 70).
Although in many respects superb, a drawback in the HECL in bioaffinity assays is the need of long incubation time in order to get the reacting molecules into equilibrium, which is necessary to optimize the analytical accuracy. Later, it was found out that a significant improvement in performance could be acquired by placing a thin porous film on the work electrode, and by producing CIPF-devices (US2009178924 (A1), Ala-Kleme et al.).
In conventional electrochemistry, electrodes are some times integrated on the same plane, but theoretically, this should not work while using hot electron electrochemistry, since the HECL should be emitted only in the outer edges of the working electrode (cathode) closest to the counter electrode. However, while testing we found out that, for some reasons in the electrolytic cell having sufficiently high volume of electrolyte solution the HECL is emitted evenly over the whole working electrode surface even if the counter electrodes are situated in the same plane on an electrode chip (integrated electrode chips, IEChip) normally made of insulating materials such as glass, ceramics or organic polymers.
Labmaster Ltd (Turku, Finland) has worked with their diagnostics strip for almost ten years and the solution developed by them is a rather simple device containing a single piece of oxide-coated silicon embedded in plastics and an essential multipurpose membrane to input samples and reagents for the bioaffinity assays (US2009178924, Ala-Kleme et al.). The major drawback of these strips is that all the measurements are carried out in an instrument's cell which has to be very carefully washed and cleaned between each measurement to avoid carry-over and the deterioration of the counter electrode built in the instrument is also problematic.
Recently, means to construct truly disposable cartridges with extremely accurate and reproducible electrodes which naturally also provide highly accurate results in practical analysis have been disclosed by us (FI 20100246, S. Kulmala et al., FI 20100251, S. Kulmala et al. and FI 20100253, S. Kulmala et al.). The basis of these inventions is either the use of Integrated Electrode Chips (IEChips) containing an anode and a cathode integrated on the same plane for the use in HECL detection, or Electrode/Electrode—chips (EEChips) including an electrode pair for any polarity made of carbon paste and usable in evoking EL of lanthanide chelate labels. The IEChips and EEChips are mainly used in disposable bioaffinity cartridges such as in cartridges for immunoanalysis or DNA-probing. IEChips and EEChips are called from now on with a combined name, electrode chip, EChip.
Quite early, we could see that, if an optically transparent working electrode and also an optically transparent counter electrode is used in HECL (M. Flakansson et al., Anal. Chim. Acta 541 (2005) 137-141.), two analytes can be determined. However, it is too expensive to include two photon counting detectors on a single instrument and a much better solution, if an electrolytical cell is invented which allows the use of only a single light detection unit. The problem of the recent inventions is that only one analyte (FI 20100246, S. Kulmala et al., FI 20100251, S. Kulmala et al.) or two analytes (FI 20100253, S. Kulmala et al.) can be determined when a single light detector is used. The present invention discloses how novel multipurpose EChips can be fabricated and used in analysis, either in multi analyte determinations, or in single analyte determinations using internal standards or standard additions or other types of referencing methods (FI 20100248, S. Kulmala et al). The priority is claimed on the basis of the applications FI 20100248, S. Kulmala et al.; FI 20100246, S. Kulmala et al.; FI 20100251, S. Kulmala et al. or FI 20100253, S. Kulmala et al. in the event that any of those is considered to prevent the novelty of the present invention.
According to the present invention, the EChips for multipurpose use can be constructed, and also a significant improvement in the use of CIPF-devices (US2009178924 (A1), Ala-Kleme et al.) is achieved utilizing disposable HECL and EL cartridges containing the above-mentioned EChips as described in the claims 1-10.